[Down-regulated clusterin expression enhances sensitivity of hepatoma cells to anti-cancer drugs].

OBJECTIVE: To investigate the relationship between and underlying mechanistic pathway of clusterin (CLU) and chemo-resistance ofhepatocellular carcinoma (HCC) cells. METHODS: CLU protein expression in HCC cell lines (Hep3B, SMMC7721, PLC, and HepG2) and HepG2/ADM cells was quantified by western blotting. Four short-hairpin (sh)RNAs designed to block CLU-mRNA were generated, screened by RT-PCR, and transfected into the cells to determine effects of CLU on cell viability and apoptosis. Effects of CLU blockade on drug efflux pump activity were measured by flow cytometry. RESULTS: CLU was found to be over-expressed in HCC cell lines and HepG2/ADM cells. The four shRNAs inhibited CLU-mRNA as follows (vs. levels in untransfected cells): shRNA-1: 73.68% (q =23.011, P < 0.01), shRNA-2: 39.26% (q =11.991, P < 0.01), shRNA-3: 62.36% (q =19.392, P < 0.01), and shRNA-4: 55.35% (q =17.149, P < 0.01). shRNA-mediated depletion of CLU led to increased sensitivity to anti-cancer drugs and increased doxorubicin-induced apoptosis in HepG2/ADM cells, as evidenced by the apoptosis ratio of the shRNA-1 group of 39.28% vs. the apoptosis ratio of the untransfected control group of 4.92%. Silencing of CLU also decreased drug etflux pump activity, and the level of MDR1/P-gp expression was significantly reduced (shRNA-1 group vs.untransfected control group: q =14.604, P < 0.01). CONCLUSION: CLU repression may enhance sensitivity of HCC cells to anti-cancers drugs and represents a potential molecular-target for reversal of multidrug-resistant HCC.

Mitochondrial carnitine palmitoyl transferase-II inactivity aggravates lipid accumulation in rat hepatocarcinogenesis.

AIM: To investigate the dynamic alteration of mitochondrial carnitine palmitoyl transferase II (CPT-II) expression during malignant transformation of rat hepatocytes. METHODS: Sprague-Dawley male rats were fed with normal, high fat (HF), and HF containing 2-fluorenylacetamide (2-FAA) diet, respectively. According to the Hematoxylin and Eosin staining of livers, rats were divided into control, fatty liver, degeneration, precancerous, and cancerous groups. Liver lipids were dyed with Oil Red O, CPT-II alterations were analyzed by immunohistochemistry, and compared with CPT-II specific concentration (µg/mg protein). Levels of total cholesterol (Tch), triglyceride (TG), and amino-transferases [alanine aminotransferase (ALT), aspartate aminotransferase (AST)] were determined by the routine methods. RESULTS: After intake of HF and/or HF+2-FAA diets, the rat livers showed mass lipid accumulation. The lipid level in the control group was significantly lower than that in other groups. The changes of serum TG and Tch levels were abnormally increasing, 2-3 times more than those in the controls (P < 0.05). During the rat liver morphological changes from normal to cancer development process with hepatocyte injury, serum AST and ALT levels were significantly higher (4-8 times, P < 0.05) than those in the control group. The specific concentration of CPT-II in liver tissues progressively decreased during hepatocyte malignant transformation, with the lowest CPT-II levels in the cancer group than in any of the other groups (P < 0.05). CONCLUSION: Low CPT-II expression might lead to abnormal hepatic lipid accumulation, which should promote the malignant transformation of hepatocytes.

Oncogenic Wnt3a expression as an estimable prognostic marker for hepatocellular carcinoma.

AIM: To investigate member 3a of Wingless-type MMTV integration site family (Wnt3a) expression in cancerous and surrounding tissues and the relationship between clinicopathologic features of hepatocellular carcinoma (HCC) and Wnt3a expression. METHODS: Wnt3a expression and cellular distribution and clinicopathologic characteristics in cancerous tissue and matched surrounding tissues were analyzed in 80 HCC patients from January 2006 to August 2008 by tissue microarrays and immunohistochemistry. The overall and disease-free survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models. RESULTS: The incidence of oncogenic Wnt3a expression in the cancerous group was up to 96.25% (77 of 80), which was significantly higher (χ(2) = 48.818, P < 0.001) than that in the surrounding group (46.25%, 37 of 80). Brown Wnt3a staining gradually increased with clinical staging that showed very strong staining in advanced HCC. The clinicopathologic features of high Wnt3a expression in HCC were related to poorly-differentiated grade (χ(2) = 20.211, P < 0.001), liver cirrhosis (χ(2) = 8.467, P < 0.004), hepatitis B virus (HBV) infection (χ(2) = 12.957, P < 0.001), higher tumor-node-metastasis stage (χ(2) = 22.960, P < 0.001), and 5-year survival rate (χ(2) = 15.469, P < 0.001). CONCLUSION: Oncogenic Wnt3a expression associated with HBV infection and cirrhotic liver might be an independent prognostic factor for HCC.

Reversal of multidrug resistance of hepatocellular carcinoma cells by metformin through inhibiting NF-κB gene transcription.

AIM: To interfere with the activation of nuclear factor-κB (NF-κB) with metformin and explore its effect in reversing multidrug resistance (MDR) of hepatocellular carcinoma (HCC) cells. METHODS: Expression of P-glycoprotein (P-gp) and NF-κB in human HepG2 or HepG2/adriamycin (ADM) cells treated with pCMV-NF-κB-small interference RNA (siRNA) with or without metformin, was analyzed by Western blot or fluorescence quantitative PCR. Cell viability was tested by CCK-8 assay. Cell cycle and apoptosis were measured by flow cytometry and Annexin-V-PE/7-AnnexinV apoptosis detection double staining assay, respectively. RESULTS: P-gp overexpression in HepG2 and HepG2/ADM cells was closely related to mdr1 mRNA (3.310 ± 0.154) and NF-κB mRNA (2.580 ± 0.040) expression. NF-κB gene transcription was inhibited by specific siRNA with significant down-regulation of P-gp and enhanced HCC cell chemosensitivity to doxorubicin. After pretreatment with metformin, HepG2/ADM cells were sensitized to doxorubicin and P-gp was decreased through the NF-κB signaling pathway. The synergistic effect of metformin and NF-κB siRNA were found in HepG2/ADM cells with regard to proliferation inhibition, cell cycle arrest and inducing cell apoptosis. CONCLUSION: Metformin via silencing NF-κB signaling could effectively reverse MDR of HCC cells by down-regulating MDR1/P-gp expression.

A new method for the determination of biological oxygen demand in domestic wastewater by headspace gas chromatography.

A headspace gas chromatographic method (HS-GC) has been developed for the determination of biological oxygen demand (BOD) in the water samples from domestic wastewater treatment plants. The method is based on measuring the remaining oxygen in the headspace of the sample (that has been seeded with microorganisms) in a closed container after a period of incubation. The relative standard deviation of the method in replicate testing was <4.9%. Further, the results differed by less than 6% when compared with the widely used reference method (the ISO standard) for determining BOD5. The limit of quantification in BODn testing was about 1.8 mg/L. The new method is simple and suitable for use in the batch sample testing for BODn measurement.